Pathogenesis Quantification Systems and Treatment Methods for Citrus Greening Blight

ABSTRACT

The invention relates to a novel pathogenesis model, method, or kit for detecting pathogenesis in a subject. In particular, the invention provides a pathogen index derived from a ratio of the amount of dual pathogen targets relative to the amount of host quantitative measures. The pathogen index is used in diagnosis, prognosis, and/or treatment strategy of any disease, including citrus greening diseases (HLB). Research tools and methods for screening drugs for treating or preventing citrus greening diseases, as well as treatment or prevention methods for citrus greening diseases are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT Application No.PCT/US2014/063181 filed Oct. 30, 2014, which claims priority to U.S.Provisional Application Ser. No. 61/897,626, filed Oct. 30, 2013,entitled “Pathogenesis Quantification Systems and Treatment Methods forCitrus Greening Blight”, the entire contents of which are incorporatedby reference herein.

FIELD OF THE INVENTION

The present invention relates particularly to detection and control ofphage and bacteria that are pathogenic to host plants and mammals.

The present invention is exemplified in the context of the citrusgreening blight, also known as Huanglongbing (HLB), and more generallyrelates to testing and treatment methods and compositions based ondetermination of a pathogenic index from a dual pathogen: multiplex hostquantification ratio or quartile of pathogenesis designed specificallyfor diagnosis and treatment of quartiles and secondarily for drugdevelopment, and as a research tool for the identification of drugscandidates.

BACKGROUND OF THE INVENTION

Citrus greening blight, also known as Huanglongbing (HLB), is a lethaldisease in citrus plants across the world, causing significant fruitloss and death of infected trees. HLB infection affects the leaves,twigs and fruit of the tree, eventually causing the whole tree todecline. Symptoms of HLB include blotchy, mottled and yellow-veinedleaves, lopsided and small, green, salty and bitter flavored fruit, twigdieback, and overall tree stunting and decline. Diagnosis of HLB iscomplicated by the fact that some of the symptoms of HLB are similar tosymptoms of other tree ailments, including nutritional deficiency. HLBcontrol often requires destroying infected trees, which may not evenshow symptoms of HLB for several years after becoming infected.

Polymerase chain reaction diagnostic testing has identified the majorpathogen associated with symptoms of HLB as the bacterium CandidatusLiberibacter spp, including the Ca. L. asiaticus (LAS), Ca. L. africanusand Ca. L. americanus species. The bacteria is believed to impairnutrient transfer in a citrus tree's phloem and leaf stems, wherein theplant pathology is suspected to be caused by nutrient deficiency.Antibiotic control of the putative causative bacterium is a passivesolution but an expensive means of grove management that couldnever be aplausible solution. Low replication levels of Ca. L. in citrus requirehigh sensitivity methods to detect their presence. PCR based methodsthat have been employed for the detection of Ca. L. asiaticus bacteriuminclude qualitative PCR assays (Saponari et al., 2013, J. Virol.Methods. 193(2): 478-86) and tissue-blot diagnosis (Nageswara-Rao M. etal., 2013, Mol. Cell Probes. 27(5-6): 176-83), each of which are hereinincorporated by reference.

Bacteriophages or phages are viruses which infect bacteria. The majorityare DNA viruses. Each phage attacks only particular species or in manycases only certain strain within the species. Structurally, abacteriophage or phage is composed of a polygonal head consisting of DNAsurrounded by a thin protein membrane and a short straight tailconsisting of protein. However, spherical and filamentous phages havealso been reported. The life cycle of bacteriophages includes twocycles: vegetative cycle and lysogenic cycle. In the vegetative cycle,the phage is called virulent phage, and the cycle contains the steps ofattachment (adsorption), penetration, eclipse phase, replication,assembly, and release. In the lysogenic cycle, the phage is calledtemperate phage, and the cycle contains the steps of attachment(adsorption), penetration, eclipse phase, and then unlike the virulentphage in the vegetative cycle, the temperate phage after the eclipsephase does not replicate, instead, it is integrated into the bacterialchromosome and remains latent for a period of time. The integrated phageis now called prophage, and the bacteria carrying prophage is calledlysogen. The lysogen is not lysed but grows and multiplies with prophageas a part of the chromosome. The lysogen can acquire new properties, andthe prophage may revert to virulent phage and infected bacteria may belysed. The practical applications of bacteriophages include, but are notlimited to, bacteriophage typing, classification of bacteria into phagegroups, phage lysogenic conversion, phage therapy, and as cloningvectors.

Bacteriophages such as those associated with Staphylococci and theintestinal commensal bacterium E. faecalis are deemed important factorsin the life cycle of host bacteria that may affect the pathogenicity ofthe host (Deghorain and Melderen, 2012, Viruses 4:3316-35; Duerkop etal., PNAS 109(43):17621-626). Analysis of the Ca. L. asiaticus genomederived from HLB symptomatic citrus has revealed the presence of twocircular phage genomes, SC1 and SC2 (Zhang et al., 2011, MolecularPlant-Microbe Interactions 24(4):458-68). Three DNA fragments (In-2.6,In-1.0 and In-0.6) of the non-cultured, bacterial-like organism (BLO)associated with citrus greening disease were cloned, and nucleotidesequence determination showed that the genome of the non-cultured BLOassociated with citrus greening disease contains the nusG-rp1KAJL-rpoBCgene cluster and the gene for a bacteriophage type DNA polymerase(Villechanoux et al., 1993, Curr Microbiol. 26(3):161-6), the entirecontents of this reference is incorporated by reference herein. Completegenome of Liberibacter crescens BT-1 was sequenced and contains moregenes in thiamine and essential amino acid biosynthesis, as well ascontains two prophage regions (Leonard et al., 2012, Standards inGenomic Sciences 7:271-83), the entire contents of which is incorporatedby reference herein.

In light of the foregoing, there still remains a need in the art for HLBdiagnosis methods and eradication methods and compositions for thedisease, as well as more generally diagnostic and treatment methods andcompositions for pathogen-pathogenic-host mediated disease systems.

SUMMARY OF THE INVENTION

Various embodiments described herein provide a novel pathogenesis modelfor diagnostic, prognostic, drug screening, and treatment methods andrelated algorithms useful for detecting pathogenesis in a biologicalsample. In certain embodiments, the invention provides a method fordetecting pathogenesis in a biological sample, comprising: a)quantifying the amount of a nucleic acid in the sample specific for afirst organism that is a pathogen associated with a second organism thatin tandem these two organisms affects a host organism to determine apathogen quantitative measurement, b) quantifying the amount of anucleic acid in the sample specific for the host organism response topathogens to determine a multiplex quantitation for targets associatedwith the pathogenesis of the disease; and c) calculating the ratio ofthe amount of dual pathogen targets relative to the amount of hostquantitative measures, wherein said ratio provides a pathogen index fordetecting pathogenesis. In certain embodiments, a greater pathogen indexindicates a poorer prognosis and can be used for drug treatment efficacyand drug screening.

In certain embodiments, the quantification of the nucleic acid in thesample specific for the first organism is performed by contacting thenucleic acid from the sample with a first set of oligonucleotide primersin a nucleic acid amplification reaction, the first set ofoligonucleotides being at least partially complementary to the nucleicacid of the first organism, and wherein the quantification of thenucleic acid in the sample specific for the second organism is performedby contacting the nucleic acid from the sample with a second set ofoligonucleotide primers in a nucleic acid amplification reaction, thesecond set of oligonucleotides being at least partially complementary tothe nucleic acid of the second organism, and further comprisingdetecting amplicons from the nucleic acid amplification reactions, andwherein the quantification of the nucleic acid in the sample specificfor the third host organism is performed by contacting the nucleic acidfrom the sample with a third set of oligonucleotide primers in a nucleicacid amplification reaction, the third set of oligonucleotides being atleast partially complementary to the nucleic acid of the third organism,and further comprising detecting amplicons from the nucleic acidamplification reactions wherein the amplification reactions occur in thesame container, and calculating the ratio of the amount of pathogenratio relative to the amount of host load based on the relative numberof amplicons to derive the pathogen index or quartile. As used herein,the pathogen organism is a virus and bacteria, and the host organism isplant or animal. When the host organism is a bacteria, the host may alsoreside within or in association with another host, such as a plant oranimal, e.g., human. In certain embodiments, the pathogenesis is citrusgreen blighting or Huanglongbin (HLB), the pathogen or the firstorganism can be a bacterio-phage or virus, and the second organism isCandidatus Liberibacter asiaticus (LAS) bacteria and the host is a widearray of fruiting and non-fruiting plants. The pathogen index may alsobe derived from a further correlation with any inducer or factor genepathogenically tied together with the dual pathogen to hostrelationship.

In certain embodiments, the inventive method further comprises a reportalgorithm correlated with the pathogen index or quartile providingclinical utility as a treatment strategy. In some embodiments, theinventive method further comprises a step of quantifying the amount of anucleic acid in the sample specific for an inducer of the pathogenorganism to determine an inducer load; and wherein the pathogen indexand the inducer load are correlated with the report index fordetermining prognosis and providing clinical utility as a treatmentstrategy. The inventive method further provides that multiple uniquenucleic acids specific to the pathogen organisms, the host organism orthe inducer are quantified and correlated in the report index.

The nucleic acid quantified in the invention includes, but is notlimited to, RNA or DNA. In certain embodiments, the phage RNA and thebacterial DNA are quantified, and a ratio of phage RNA to bacterial DNAis calculated to derive a pathogen index. The nucleic acid can beamplified for relative quantitative determination by any conventionalnucleic acid detection reaction now known or later developed including,but not limited to standard polymerase chain reaction (SPCR), real-timePCR, recombinase polymerase amplification (RPA), helicase-dependentamplification (HAD), loop mediate isothermal amplification (LAMP), andnicking enzyme amplification reaction (NEAR).

In certain embodiments, the invention provides a kit for detecting apathogenesis, such as citrus greening blight (HLB), in a sample,comprising: a) at least a first set of oligonucleotides being at leastpartially complementary to a nucleic acid of a first pathogen organismin the sample that is pathogenically associated with a second organismthat in tandem these two pathogens affects a host organism to determinea pathogen quantitative measurement, b) at least a second set ofoligonucleotides being at least partially complementary to a nucleicacid of the host organism response to the pathogen of the disease, c)reagents for nucleic acid amplification reactions with the first andsecond sets of oligonucleotides; d) instructions for conducting thenucleic acid amplification reactions and detecting one or more ampliconsthereof; and e) instructions for calculating a ratio of the ampliconsand/or the amount of dual pathogen targets relative to the amount ofhost quantitative measures, wherein said ratio provides a pathogen indexfor detecting, prognosis, drug screening, and treatment strategy orefficacy for the pathogeneis, such as citrus greening blight (HLB).

The invention further provides a method for screening a candidatetherapeutic agent for inhibition or prevention of citrus greeningblight. The invention method comprises the steps of providing a cultureof Candidatus Liberibacter with a bacteriophage having a repressed phagelytic cycle; combining the culture with a candidate therapeutic agentcandidate; and detecting inhibition of growth of the CandidatusLiberibacter culture indicating that the candidate therapeutic agentinhibits or prevents citrus greening blight. In certain embodiments, thebacteriophage is a lytic phage, including but not limited to, SC1, SC2,and SC3 lytic phage, and wherein the phage lytic cycle is repressed orgenetically deleted.

The invention also provides a method for treating and/or preventingbacteriophage or viral infection in bacteria, plant or animalindividual, said method comprising administering to the individual inneed a composition comprising an effective amount of an agent selectedfrom the group consisting of nucleoside, non-nucleoside, nucleotide,non-nucleotide, ribonucleoside, and a ribonucleoside analog thatselectively inhibits viral replication. In certain embodiments, thebacteriophage infection is in a Candidatus Liberibacter in a citrus treethat results in citrus greening blight, such as HLB or LAS. In certainembodiments, an antiviral agent is used to treat and/or prevent HLB orLAS. The invention contemplates different classes of antiviral agents,now known or later developed, including but not limited to, naturalantiviral compounds, such as interferon-α (IFN-α) with maximal antiviralactivity and interferon-β (IFN-β) with intermediate activity, andsynthetic antiviral agents, which based on their point of action in theviral replication cycle, are divided into the following groups: 1)nucleoside analogs, such as acyclovir (ACV) and its pro-drugvalacyclovir, ribavirin, zidovudin, and azidothymidine (AZT), whichblock viral nucleic acid synthesis; 2) nucleotide analogs, e.g.,cidofovir (CDV), ganciclovir (GCV) (or valganciclovir) which differ fromnucleoside analogs by having an attached phosphate group. The nucleotideanalogs can persist in cells for long periods of time; 3) non-nucleosidereverse transcriptase inhibitor, e.g., nevirapine, which bind to reversetranscriptase; 4) protease inhibitors, e.g., saquinavir, which inhibitsviral protease; and 5) other types, such as amantidine and rimantidinewhich inhibit influenza viruses by inhibiting viral uncoating, and/orthe pyrophosphate analog phosphonoformic (PFA, foscarnet) which inhibitsreverse transcriptase in HIV and viral polymerase in Herpes viruses (seee.g., Bennett and Gotte, 2013, Viruses 5:54-86, the review aims todiscuss strengths, limitations, and opportunities of the phage surrogatewith emphasis placed on its utility in the discovery and development ofantiherpetic drugs), the entire contents of which is incorporated byreference herein.

In certain embodiments, the antiviral agents are ATP analogs, such as2′-deoxy-2′-azidoadenosine 5′-triphosphate (AzTP) and/or2′-deoxy-2′-fluoroadenosine 5′-triphophate (AfTP), which inhibits RNAsynthesis by E. coli RNA polymerase (Ishihama et al., 1980, J. Biochem.87: 825-30), the entire content of which is incorporated by referenceherein. In other embodiments, the antiviral agents are azidothymidine(AZT), the thymidine analog 3′-azido-3′-deoxythymidine (aka BW A509U),which has potent bacterial activity against many members of the familyEnterobacteriaceae, and the AZT-triphophate is the most potent inhibitorof replicative DNA synthesis, and/or a specific DNA chain terminator inthe in vitro DNA polymerization reaction, which may explain the lethalproperties of this compound against susceptible microorganisms (Elwellet al., 1987, Antimicrobial Agents and Chemotherapy, 31(2): 274-80), theentire contents of which is incorporated by reference herein.

In certain embodiments, the treatment method can further comprise anucleotide pool reducer, such as hydroxyurea in an effective amount. Insome embodiments, the treatment composition further comprises asurfactant or penetrant for enhancing delivery of the composition to atargeted location (such as a leaf) of the affected plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a novel pathogenesis model.

FIG. 2 illustrates normal pores in angiosperm sieve tube elements andcompanion cells.

FIG. 3 illustrates HLB flagellated in angiosperm sieve tube elements andcompanion cells.

FIG. 4 illustrates blocked pores where phage particles, bacterial cellwall, starch, remnant peptidoglycan and cellular contents are releasedsimultaneously in Lytic Phage Burst (LPB).

FIG. 5 illustrates the “Greening Effect” where downstream phloem isdried up in a “river bed” corky appearance. Transpiration is halted forthis section of tree independent of other systemic nutrient routes.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the invention is not limited in itsapplication to the details of and the arrangement of components setforth in the following description. It is also to be understood thatthis invention is not limited to particular oligonucleotide probes,methods, compositions, reaction mixtures, kits, systems, computers, orcomputer readable media, which can, of course, vary. It is further to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. Further, unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. Each of the references cited herein is incorporated byreference in its entirety. In describing and claiming the presentinvention, the following terminology and grammatical variants will beused in accordance with the definitions set forth below.

Definitions:

An “amplification reaction” refers to a primer initiated replication ofone or more target nucleic acid sequences or complements thereto.

An “amplicon” refers to a molecule made by copying or transcribinganother molecule, e.g., as occurs in transcription, cloning, and/or in atechnique such as polymerase chain reaction (“PCR”) (e.g., stranddisplacement PCR amplification (SDA), duplex PCR amplification,real-time PCR, recombinase polymerase amplification (RPA),helicase-dependent amplification (HAD), loop mediate isothermalamplification (LAMP), and nicking enzyme amplification reaction (NEAR))or other nucleic acid amplification technique. Typically, an amplicon isa copy of a selected nucleic acid (e.g., a template or target nucleicacid) or is complementary thereto.

An “amplified signal” refers to increased detectable signal that can beproduced in the absence of, or in conjunction with, an amplificationreaction. Exemplary signal amplification techniques are described in,e.g., Cao et al. (1995) “Clinical evaluation of branched DNA signalamplification for quantifying HIV type 1 in human plasma,” AIDS Res HumRetroviruses 11(3):353-361, and in U.S. Pat. No. 5,437,977 to Segev,U.S. Pat. No. 6,033,853 to Delair et al., and U.S. Pat. No. 6,180,777 toHorn, which are each incorporated by reference.

The term “attached” or “conjugated” refers to interactions and/or statesin which material or compounds are connected or otherwise joined withone another. These interactions and/or states are typically produced by,e.g., covalent bonding, ionic bonding, chemisorption, physisorption, andcombinations thereof. In certain embodiments, for example,oligonucleotide probes are attached to solid supports. In some of theseembodiments, an oligonucleotide probe is conjugated with biotin (i.e.,is biotinylated) and a solid support is conjugated with avidin such thatthe probe attaches to the solid support via the binding interaction of,e.g., biotin and avidin.

Molecular species “bind” when they associate with one another viacovalent and/or non-covalent interactions. For example, twocomplementary single-stranded nucleic acids can hybridize with oneanother to form a nucleic acid with at least one double-stranded region.To further illustrate, antibodies and corresponding antigens can alsonon-covalently associate with one another.

The term “cleavage” refers to a process of releasing a material orcompound from attachment to another material or compound. In certainembodiments, for example, oligonucleotides are cleaved from, e.g., asolid support to permit analysis of the oligonucleotides bysolution-phase methods. See, e.g., Wells et al. (1998) “Cleavage andAnalysis of Material from Single Resin Beads,” J. Org. Chem. 63:6430,which is incorporated by reference.

A “character” when used in reference to a character of a characterstring refers to a subunit of the string. In one embodiment, thecharacter of a character string encodes one subunit of an encodedbiological molecule. Thus, for example, where the encoded biologicalmolecule is a polynucleotide or oligonucleotide, a character of thestring encodes a single nucleotide.

A “character string” is any entity capable of storing sequenceinformation (e.g., the subunit structure of a biological molecule suchas the nucleotide sequence of a nucleic acid, etc.). In one embodiment,the character string can be a simple sequence of characters (letters,numbers, or other symbols) or it can be a numeric or codedrepresentation of such information in tangible or intangible (e.g.,electronic, magnetic, etc.) form. The character string need not be“linear,” but can also exist in a number of other forms, e.g., a linkedlist or other non-linear array (e.g., used as a code to generate alinear array of characters), or the like. Character strings aretypically those which encode oligonucleotide or polynucleotide strings,directly or indirectly, including any encrypted strings, or images, orarrangements of objects which can be transformed unambiguously tocharacter strings representing sequences of monomers or multimers inpolynucleotides, or the like (whether made of natural or artificialmonomers).

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

A “composition” refers to a combination of two or more differentcomponents. In certain embodiments, for example, a composition includesa solid support that comprises one or more oligonucleotide probes, e.g.,covalently or non-covalently attached to a surface of the support. Inother embodiments, a composition includes one or more oligonucleotideprobes in solution.

The term “deletion” in the context of a nucleic acid sequence refers toan alteration in which at least one nucleotide is removed from thenucleic acid sequence, e.g., from a 5′-terminus, from a 3′-terminus,and/or from an internal position of the nucleic acid sequence.

The term “derivative” refers to a chemical substance relatedstructurally to another substance, or a chemical substance that can bemade from another substance (i.e., the substance it is derived from),e.g., through chemical or enzymatic modification. To illustrate,oligonucleotide probes are optionally conjugated with biotin or a biotinderivative. To further illustrate, one nucleic acid can be “derived”from another through processes, such as chemical synthesis based onknowledge of the sequence of the other nucleic acid, amplification ofthe other nucleic acid, or the like.

The term “selectively bind” or “selective binding” in the context ofnucleic acid detection reagents refers to a nucleic acid detectionreagent that binds to one or more target nucleic acid or a substantiallyidentical variant or complement thereof to a greater extent than thenucleic acid detection reagent binds, under the same hybridizationconditions, to non-target nucleic acids. The term “detectably bind”refers to binding between at least two molecular species (e.g., a probenucleic acid and a target nucleic acid, a sequence specific antibody anda target nucleic acid, etc.) that is detectable above a backgroundsignal (e.g., noise) using one or more methods of detection. The term“selectively detect” refers to the ability to detect one or more targetnucleic acid or a substantially identical variant or complement thereofto a greater extent than non-target nucleic acids from organisms.

Nucleic acids are “extended” or “elongated” when additional nucleotides(or other analogous molecules) are incorporated into the nucleic acids.For example, a nucleic acid is optionally extended by a nucleotideincorporating biocatalyst, such as a polymerase that typically addsnucleotides at the 3′ terminal end of a nucleic acid.

An “extended primer nucleic acid” refers to a primer nucleic acid towhich one or more additional nucleotides have been added or otherwiseincorporated (e.g., covalently bonded thereto).

Nucleic acids “hybridize” or “bind” when they associate with oneanother, typically in solution. Nucleic acids hybridize due to a varietyof well characterized physio-chemical forces, such as hydrogen bonding,solvent exclusion, base stacking and the like. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes part I chapter 2, “Overview of principles ofhybridization and the strategy of nucleic acid probe assays,” (Elsevier,N.Y.), as well as in Ausubel (Ed.) Current Protocols in MolecularBiology, Volumes I, II, and III, 1997, which is incorporated byreference. Hames and Higgins (1995) Gene Probes 1 IRL Press at OxfordUniversity Press, Oxford, England, (Hames and Higgins 1) and Hames andHiggins (1995) Gene Probes 2 IRL Press at Oxford University Press,Oxford, England (Hames and Higgins 2) provide details on the synthesis,labeling, detection and quantification of DNA and RNA, includingoligonucleotides. Both Hames and Higgins 1 and 2 are incorporated byreference.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization assays or experiments, such as nucleic acid amplificationreactions, Southern and northern hybridizations, or the like, aresequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2. Forpurposes of the present invention, generally, “highly stringent”hybridization and wash conditions are selected to be at least about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the T_(m) for a particular probe.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids on a filter in a Southern or northern blotis 50% formalin with 1 mg of heparin at 42° C., with the hybridizationbeing carried out overnight. An example of stringent wash conditions isa 0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook et al., MolecularCloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001), which is incorporated byreference, for a description of SSC buffer). Often the high stringencywash is preceded by a low stringency wash to remove background probesignal. An example low stringency wash is 2×SSC at 40° C. for 15minutes. In general, a signal to noise ratio of 5× (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization.

Comparative hybridization can be used to identify nucleic acids orpreferred primers for complementary nucleic acid sets for amplificationof the invention. In particular, detection of stringent hybridization inthe context of the present invention indicates strong structuralsimilarity to, e.g., the nucleic acids of the desired bacterial, phage,virus, etc. For example, it is desirable to identify test nucleic acidsthat hybridize to the exemplar nucleic acids herein under stringentconditions. One measure of stringent hybridization is the ability todetectably hybridize to one of the desired nucleic acids (e.g., nucleicacids of bacteria and/or phage associated with HLB or other citrusgreening blight, and complements thereof) under stringent conditions.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid.

For example, in determining highly stringent hybridization and washconditions, the stringency of the hybridization and wash conditions aregradually increased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents such as formalin in the hybridizationor wash), until a selected set of criteria is met. For example, thestringency of the hybridization and wash conditions are graduallyincreased until a probe consisting of or comprising one or more desirednucleic acid sequences and complementary polynucleotide sequencesthereof, binds to a perfectly matched complementary target (e.g., anucleic acid comprising one or more nucleic acid sequences of bacteriaand/or phage associated with HLB or other citrus greening blight, andcomplementary polynucleotide sequences thereof), with a signal to noiseratio that is at least 5× as high as that observed for hybridization ofthe probe to a non-target nucleic acid. In this case, non-target nucleicacids are those from organisms other than the desired bacteria, phage,virus, etc. Such non-target nucleic acid sequences can be identified in,e.g., GenBank by one of skill in the art. A test nucleic acid is said tospecifically hybridize to a probe nucleic acid when it hybridizes atleast one-half as well to the probe as to the perfectly matchedcomplementary target, i.e., with a signal to noise ratio at leastone-half as high as hybridization of the probe to the target underconditions in which the perfectly matched probe binds to the perfectlymatched complementary target with a signal to noise ratio that is atleast about 5×-10× as high as that observed for hybridization to thenon-target nucleic acids.

Ultra high-stringency hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to the non-target nucleic acids.A target nucleic acid which hybridizes to a probe under such conditions,with a signal to noise ratio of at least one-half that of the perfectlymatched complementary target nucleic acid is said to bind to the probeunder ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the stringency of hybridization and/or washconditions of the relevant hybridization assay. For example, those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10×,20×, 50×, 100×, or 500× or more as high as that observed forhybridization to the non-target nucleic acids can be identified. Atarget nucleic acid which hybridizes to a probe under such conditions,with a signal to noise ratio of at least one-half that of the perfectlymatched complementary target nucleic acid is said to bind to the probeunder ultra-ultra-high stringency conditions.

“Selectively hybridizing” or “selective hybridization” occurs when anucleic acid sequence hybridizes to a specified nucleic acid targetsequence to a detectably greater degree than its hybridization tonon-target nucleic acid sequences. Selectively hybridizing sequenceshave at least 50%, or 60%, or 70%, or 80%, or 90% sequence identity ormore, e.g., typically 95-100% sequence identity (i.e., complementarity)with each other.

A “sequence” of a nucleic acid refers to the order and identity ofnucleotides in the nucleic acid. A sequence is typically read in the 5′to 3′ direction. The terms “identical” or percent “identity” in thecontext of two or more nucleic acid or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence, e.g., asmeasured using one of the sequence comparison algorithms available topersons of skill or by visual inspection. Exemplary algorithms that aresuitable for determining percent sequence identity and sequencesimilarity are the BLAST programs, which are described in, e.g.,Altschul et al. (1990) “Basic local alignment search tool” J. Mol. Biol.215:403-410, Gish et al. (1993) “Identification of protein codingregions by database similarity search” Nature Genet. 3:266-272, Maddenet al. (1996) “Applications of network BLAST server” Meth. Enzymol.266:131-141, Altschul et al. (1997) ““Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs” Nucleic Acids Res.25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A new network BLASTapplication for interactive or automated sequence analysis andannotation” Genome Res. 7:649-656, which are each incorporated byreference. Many other optimal alignment algorithms are also known in theart and are optionally utilized to determine percent sequence identity.

The phrase “in solution” refers to an assay or reaction condition inwhich the components of the assay or reaction are not attached to asolid support and are present in a liquid medium. Exemplary liquidmediums include aqueous and organic fluids. For example, certain assaysof the invention include incubating oligonucleotide probes together withCandidatus Liberibacter asiaticus (LAS or HLB) bacteria nucleic acidsand LAS nucleic acid amplicons in solution to allow hybridization tooccur.

The term “insertion” in the context of a nucleic acid sequence refers toan alteration in which at least one nucleotide is added to the nucleicacid sequence, e.g., at a 5′-terminus, at a 3′-terminus, and/or at aninternal position of the nucleic acid sequence.

A “label” refers to a moiety attached (covalently or non-covalently), orcapable of being attached, to a molecule, which moiety provides or iscapable of providing information about the molecule (e.g., descriptive,identifying, etc. information about the molecule) or another moleculewith which the labeled molecule interacts (e.g., hybridizes, etc.).Exemplary labels include fluorescent labels (including, e.g., quenchersor absorbers), weakly fluorescent labels, non-fluorescent labels,colorimetric labels, chemiluminescent labels, bioluminescent labels,radioactive labels, mass-modifying groups, antibodies, antigens, biotin,haptens, enzymes (including, e.g., peroxidase, phosphatase, etc.), andthe like.

A “linker” refers to a chemical moiety that covalently or non-covalentlyattaches a compound or substituent group to another moiety, e.g., anucleic acid, an oligonucleotide probe, a primer nucleic acid, anamplicon, a solid support, or the like. For example, linkers areoptionally used to attach oligonucleotide probes to a solid support(e.g., in a linear or other logic probe array). To further illustrate, alinker optionally attaches a label (e.g., a fluorescent dye, aradioisotope, etc.) to an oligonucleotide probe, a primer nucleic acid,or the like. Linkers are typically at least bifunctional chemicalmoieties and in certain embodiments, they comprise cleavableattachments, which can be cleaved by, e.g., heat, an enzyme, a chemicalagent, electromagnetic radiation, etc. to release materials or compoundsfrom, e.g., a solid support. A careful choice of linker allows cleavageto be performed under appropriate conditions compatible with thestability of the compound and assay method. Generally a linker has nospecific biological activity other than to, e.g., join chemical speciestogether or to preserve some minimum distance or other spatialrelationship between such species. However, the constituents of a linkermay be selected to influence some property of the linked chemicalspecies such as three-dimensional conformation, net charge,hydrophobicity, etc. Exemplary linkers include, e.g., oligopeptides,oligonucleotides, oligopolyamides, oligoethyleneglycerols,oligoacrylamides, alkyl chains, or the like. Additional description oflinker molecules is provided in, e.g., Hermanson, BioconjugateTechniques, Elsevier Science (1996), Lyttle et al. (1996) Nucleic AcidsRes. 24(14):2793, Shchepino et al. (2001) Nucleosides, Nucleotides, &Nucleic Acids 20:369, Doronina et al (2001) Nucleosides, Nucleotides, &Nucleic Acids 20:1007, Trawick et al. (2001) Bioconjugate Chem. 12:900,Olejnik et al. (1998) Methods in Enzymology 291:135, and Pljevaljcic etal. (2003) J. Am. Chem. Soc. 125(12):3486, all of which are incorporatedby reference.

A “mixture” refers to a combination of two or more different components.A “reaction mixture” refers a mixture that comprises molecules that canparticipate in and/or facilitate a given reaction. An “amplificationreaction mixture” refers to a solution containing reagents necessary tocarry out an amplification reaction, and typically contains primers, athermostable DNA polymerase, dNTP's, and a divalent metal cation in asuitable buffer. A reaction mixture is referred to as complete if itcontains all reagents necessary to carry out the reaction, andincomplete if it contains only a subset of the necessary reagents. Itwill be understood by one of skill in the art that reaction componentsare routinely stored as separate solutions, each containing a subset ofthe total components, for reasons of convenience, storage stability, orto allow for application-dependent adjustment of the componentconcentrations, and, that reaction components are combined prior to thereaction to create a complete reaction mixture. Furthermore, it will beunderstood by one of skill in the art that reaction components arepackaged separately for commercialization and that useful commercialkits may contain any subset of the reaction components, which includesthe modified primers of the invention.

A “modified primer nucleic acid” refers to a primer nucleic acid thatcomprises a moiety or sequence of nucleotides that provides a desiredproperty to the primer nucleic acid. In certain embodiments, forexample, modified primer nucleic acids comprise “nucleic acidamplification specificity altering modifications” that, e.g., reducenon-specific nucleic acid amplification (e.g., minimize primer dimerformation or the like), increase the yield of an intended targetamplicon, and/or the like. Examples of nucleic acid amplificationspecificity altering modifications are described in, e.g., U.S. Pat. No.6,001,611, which is incorporated by reference. Other exemplary primernucleic acid modifications include a “restriction site linkermodification” in which a nucleotide sequence comprising a selectedrestriction site is attached, e.g., at 5′-terminus of a primer nucleicacid. Restriction site linkers are typically attached to primer nucleicacids to facilitate subsequent amplicon cloning or the like.

A “moiety” or “group” refers to one of the portions into whichsomething, such as a molecule, is divided (e.g., a functional group,substituent group, or the like). For example, an oligonucleotide probeoptionally comprises a quencher moiety, a labeling moiety, or the like.

The term “Candidatus Liberibacter asiaticus,” “Candidatus Liberibacter,”or “liberobacter” refers to a genus of gram-native bacteria in theRhizobiaceae family. Detection of the liberibacters can be based on PCRamplification of their 16S rRNA gene with specific primers known in theart or designed for this purpose. Members of the genus are plantpathogens mostly transmitted by psyllids. PCR based methods that havebeen employed for the detection of Ca. L. asiaticus bacterium includemultiplex quantitative real-time PCR assays (Saponari et al.) andtissue-blot diagnosis (Nageswara-Rao M. et al), which are each cited infull above and hereby incorporated by reference.

A term “phage” or “bacteriophage” or “bacterial virus” refers to any ofa group of viruses that infects and replicates within bacteria.Bacteriophages are composed of proteins that encapsulate a DNA or RNAgenome. Their genomes may encode as few as four genes, and as many ashundreds of genes. Phage replicate within bacteria following theinjection of their genome into the cytoplasm. Phages are widelydistributed in locations populated by bacterial hosts. Phages are seenas a possible therapy against many bacteria. A “virus” is a smallinfectious agent that replicates only inside the living cells of otherorganisms. Viruses can infect all types of life forms, from animal andplants to bacteria and archaea.

During infection a phage attaches to a bacterium and inserts its geneticmaterial into the cell. After this a phage follows one of two lifecycles, lytic (virulent or viral reproduction) or lysogenic (temperate).Lytic phages take over the machinery of the cell to make phagecomponents. They then destroy, or lyse, the cell, releasing new phageparticles. Lysogenic phages incorporate their nucleic acid into thechromosome of the host cell and replicate with it as a unit withoutdestroying the cell. Under certain conditions lysogenic phages can beinduced to follow a lytic cycle. A key difference between the lytic andlysogenic phage cycles is that in the lytic phage, the viral DNA existsas a separate molecule within the bacterial cell, and replicatesseparately from the host bacterial DNA. The location of viral DNA in thelysogenic phage cycle is within the host DNA, therefore in both casesthe virus/phage replicates using the host DNA machinery, but in thelytic phage cycle, the phage is a free floating separate molecule to thehost DNA.

The term “Candidatus Liberibacter nucleic acid” or “CandidatusLiberibacter asiaticus nucleic acid” or “bacteria or phage or virusnucleic acid” refers to a nucleic acid (and/or an amplicon thereof) thatis derived or isolated from Candidatus Liberibacter or CandidatusLiberibacter asiaticus or target bacteria, phage, or virus, etc.

The term “nucleic acid” refers to nucleotides (e.g., ribonucleotides,deoxyribonucleotides, dideoxynucleotides, etc.) and polymers thatcomprise such nucleotides covalently linked together, either in a linearor branched fashion. Exemplary nucleic acids include deoxyribonucleoicacids (DNAs), ribonucleic acids (RNAs), DNA-RNA hybrids,oligonucleotides, polynucleotides, genes, cDNAs, aptamers, antisensenucleic acids, interfering RNAs (RNAis), molecular beacons, nucleic acidprobes, peptide nucleic acids (PNAs), locked nucleic acids (LNA™),PNA-DNA conjugates, PNA-RNA conjugates, LNA™-DNA conjugates, LNA™-RNAconjugates, etc.

A nucleic acid is typically single-stranded or double-stranded and willgenerally contain phosphodiester bonds, although in some cases, asoutlined, herein, nucleic acid analogs are included that may havealternate backbones, including, for example and without limitation,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925 andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81:579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805; Letsinger et al.(1988) J. Am. Chem. Soc. 110:4470; and Pauwels et al. (1986) ChemicaScripta 26: 1419, which are each incorporated by reference),phosphorothioate (Mag et al. (1991) Nucleic Acids Res. 19:1437; and U.S.Pat. No. 5,644,048, which are both incorporated by reference),phosphorodithioate (Briu et al. (1989) J. Am. Chem. Soc. 111:2321, whichis incorporated by reference), O-methylphosphoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press (1992), which is incorporated by reference), andpeptide nucleic acid backbones and linkages (see, Egholm (1992) J. Am.Chem. Soc. 114:1895; Meier et al. (1992) Chem. Int. Ed. Engl. 31:1008;Nielsen (1993) Nature 365:566; and Carlsson et al. (1996) Nature380:207, which are each incorporated by reference). Other analog nucleicacids include those with positively charged backbones (Denpcy et al.(1995) Proc. Natl. Acad. Sci. USA 92:6097, which is incorporated byreference); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684,5,602,240, 5,216,141 and 4,469,863; Angew (1991) Chem. Intl. Ed. English30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470; Letsingeret al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and 3, ASCSymposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghvi and P. Dan Cook; Mesmaeker et al. (1994)Bioorganic & Medicinal Chem: Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; and Tetrahedron Lett. 37:743 (1996), which areeach incorporated by reference) and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters6 and 7, ASC Symposium Series 580, Carbohydrate Modifications inAntisense Research, Ed. Y. S. Sanghvi and P. Dan Cook, which referencesare each incorporated by reference. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al. (1995) Chem. Soc. Rev. pp 169-176, which isincorporated by reference). Several nucleic acid analogs are alsodescribed in, e.g., Rawls, C & E News Jun. 2, 1997 page 35, which isincorporated by reference. These modifications of the ribose-phosphatebackbone may be done to facilitate the addition of additional moietiessuch as labels, or to alter the stability and half-life of suchmolecules in physiological environments.

In addition to these naturally occurring heterocyclic bases that aretypically found in nucleic acids (e.g., adenine, guanine, thymine,cytosine, and uracil), nucleic acid analogs also include those havingnon-naturally occurring heterocyclic or modified bases, many of whichare described, or otherwise referred to, herein. In particular, manynon-naturally occurring bases are described further in, e.g., Seela etal. (1991) Hely. Chim. Acta 74:1790, Grein et al. (1994) Bioorg. Med.Chem. Lett. 4:971-976, and Seela et al. (1999) Hely. Chim. Acta 82:1640,which are each incorporated by reference. To further illustrate, certainbases used in nucleotides that act as melting temperature (TO modifiersare optionally included. For example, some of these include7-deazapurines (e.g., 7-deazaguanine, 7-deazaadenine, etc.),pyrazolo[3,4-d]pyrimidines, propynyl-dN (e.g., propynyl-dU, propynyl-dC,etc.), and the like. See, e.g., U.S. Pat. No. 5,990,303, entitled“SYNTHESIS OF 7-DEAZA-2′-DEOXYGUANOSINE NUCLEOTIDES,” which issued Nov.23, 1999 to Seela, which is incorporated by reference. Otherrepresentative heterocyclic bases include, e.g., hypoxanthine, inosine,xanthine; 8-aza derivatives of 2-aminopurine, 2,6-diaminopurine,2-amino-6-chloropurine, hypoxanthine, inosine and xanthine;7-deaza-8-aza derivatives of adenine, guanine, 2-aminopurine,2,6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine andxanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine;5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynylcytosine;5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil;5-bromouracil; 5-trifluoromethyluracil; 5-methoxymethyluracil;5-ethynyluracil; 5-propynyluracil, and the like.

Examples of modified bases and nucleotides are also described in, e.g.,U.S. Pat. No. 5,484,908, entitled “OLIGONUCLEOTIDES CONTAINING5-PROPYNYL PYRIMIDINES,” issued Jan. 16, 1996 to Froehler et al., U.S.Pat. No. 5,645,985, entitled “ENHANCED TRIPLE-HELIX AND DOUBLE-HELIXFORMATION WITH OLIGOMERS CONTAINING MODIFIED PYRIMIDINES,” issued Jul.8, 1997 to Froehler et al., U.S. Pat. No. 5,830,653, entitled “METHODSOF USING OLIGOMERS CONTAINING MODIFIED PYRIMIDINES,” issued Nov. 3, 1998to Froehler et al., U.S. Pat. No. 6,639,059, entitled “SYNTHESIS OF[2.2.1]BICYCLO NUCLEOSIDES,” issued Oct. 28, 2003 to Kochkine et al.,U.S. Pat. No. 6,303,315, entitled “ONE STEP SAMPLE PREPARATION ANDDETECTION OF NUCLEIC ACIDS IN COMPLEX BIOLOGICAL SAMPLES,” issued Oct.16, 2001 to Skouv, and U.S. Pat. Application Pub. No. 2003/0092905,entitled “SYNTHESIS OF [2.2.1]BICYCLO NUCLEOSIDES,” by Kochkine et al.that published May 15, 2003, which are each incorporated by reference.

The term “nucleic acid detection reagent” refers to a reagent thatdetectably binds (e.g., hydrogen bonds in nucleic acid hybridization, inantibody-antigen recognition, or the like, or other types of bindinginteractions) to a nucleic acid of a desired bacteria, phage, virus,etc. that associated with citrus greening diseases or HLB, asubstantially identical variant thereof in which the variant has atleast 50%, 60%, 70%, 80%, 90%, 95%, 99% sequence identity to one of saidnucleic acids or the variant. A “nucleotide” refers to an ester of anucleoside, e.g., a phosphate ester of a nucleoside. For example, anucleotide can include 1, 2, 3, or more phosphate groups covalentlylinked to a 5′ position of a sugar moiety of the nucleoside.

A “nucleotide incorporating biocatalyst” refers to a catalyst thatcatalyzes the incorporation of nucleotides into a nucleic acid.Nucleotide incorporating biocatalysts are typically enzymes. An “enzyme”is a protein-and/or nucleic acid-based catalyst that acts to reduce theactivation energy of a chemical reaction involving other compounds or“substrates.” A “nucleotide incorporating enzyme” refers to an enzymethat catalyzes the incorporation of nucleotides into a nucleic acid,e.g., during nucleic acid amplification or the like. Exemplarynucleotide incorporating enzymes include, e.g., polymerases, terminaltransferases, reverse transcriptases, telomerases, polynucleotidephosphorylases, and the like.

An “oligonucleotide” refers to a nucleic acid that includes at least twonucleic acid monomer units (e.g., nucleotides), typically more thanthree monomer units, and more typically greater than ten monomer units.The exact size of an oligonucleotide generally depends on variousfactors, including the ultimate function or use of the oligonucleotide.Oligonucleotides are optionally prepared by any suitable method,including, but not limited to, isolation of an existing or naturalsequence, DNA replication or amplification, reverse transcription,cloning and restriction digestion of appropriate sequences, or directchemical synthesis by a method such as the phosphotriester method ofNarang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester methodof Brown et al. (1979) Meth. Enzymol. 68:109-151; thediethylphosphoramidite method of Beaucage et al. (1981) TetrahedronLett. 22:1859-1862; the triester method of Matteucci et al. (1981) J.Am. Chem. Soc. 103:3185-3191; automated synthesis methods; or the solidsupport method of U.S. Pat. No. 4,458,066, or other methods known in theart. All of these references are incorporated by reference.

An oligonucleotide probe is “specific” for a target sequence if thenumber of mismatches presents between the oligonucleotide and the targetsequence is less than the number of mismatches present between theoligonucleotide and non-target sequences that might be present in asample. Hybridization conditions can be chosen under which stableduplexes are formed only if the number of mismatches present is no morethan the number of mismatches present between the oligonucleotide andthe target sequence. Under such conditions, the target-specificoligonucleotide can form a stable duplex only with a target sequence.Thus, the use of target-specific primers under suitably stringentamplification conditions enables the specific amplification of thosesequences, which contain the target primer binding sites. Similarly, theuse of target-specific probes under suitably stringent hybridizationconditions enables the detection of a specific target sequence.

The term “oligonucleotide probe,” “probe nucleic acid,” or “probe”refers to a labeled or unlabeled oligonucleotide capable of selectivelyhybridizing to a target nucleic acid under suitable conditions.Typically, a probe is sufficiently complementary to a specific targetsequence (e.g., a Candidatus Liberibacter nucleic acid or the variant)contained in a nucleic acid sample to form a stable hybridization duplexwith the target sequence under a selected hybridization condition, suchas, but not limited to, a stringent hybridization condition. Ahybridization assay carried out using the probe under sufficientlystringent hybridization conditions permits the selective detection of aspecific target sequence. The term “hybridizing region” refers to thatregion of a nucleic acid that is exactly or substantially complementaryto, and therefore hybridizes to, the target sequence. For use in ahybridization assay for the discrimination of single nucleotidedifferences in sequence, the hybridizing region is typically from about8 to about 100 nucleotides in length. Although the hybridizing regiongenerally refers to the entire oligonucleotide, the probe may includeadditional nucleotide sequences that function, for example, as linkerbinding sites to provide a site for attaching the probe sequence to asolid support or the like. In certain embodiments, an oligonucleotideprobe of the invention comprises one or more labels (e.g., a reporterdye, a quencher moiety, etc.), such as a FRET probe, a molecular beacon,or the like, which can also be utilized to detect hybridization betweenthe probe and target nucleic acids in a sample. In some embodiments, thehybridizing region of the oligonucleotide probe is completelycomplementary to the target sequence. However, in general, completecomplementarity is not necessary; stable duplexes may contain mismatchedbases or unmatched bases. Modification of the stringent conditions maybe necessary to permit a stable hybridization duplex with one or morebase pair mismatches or unmatched bases. Sambrook et al., MolecularCloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (2001), which is incorporated byreference, provides guidance for suitable modification. Stability of thetarget/probe duplex depends on a number of variables including length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, temperature, and ionic conditions. One of skill in theart will recognize that, in general, the exact complement of a givenprobe is similarly useful as a probe. One of skill in the art will alsorecognize that, in certain embodiments, probe nucleic acids can also beused as primer nucleic acids.

A “primer nucleic acid” or “primer” is a nucleic acid that can hybridizeto a template nucleic acid (e.g., a Candidatus Liberibacter nucleicacid, a substantially identical variant thereof in which the variant hasat least 50%, 60%, 70%, 80%, 90%, 95%, 99% sequence identity to one ofsaid nucleic acids or the variant, etc.) and permit chain extension orelongation using, e.g., a nucleotide incorporating biocatalyst, such asa polymerase under appropriate reaction conditions. A primer nucleicacid is typically a natural or synthetic oligonucleotide (e.g., asingle-stranded oligodeoxyribonucleotide, etc.). Although other primernucleic acid lengths are optionally utilized, they typically comprisehybridizing regions that range from about 8 to about 100 nucleotides inlength. Short primer nucleic acids generally utilize cooler temperaturesto form sufficiently stable hybrid complexes with a template nucleicacid. A primer nucleic acid that is at least partially complementary toa subsequence of a template nucleic acid is typically sufficient tohybridize with the template for extension to occur.

A primer nucleic acid can be labeled, if desired, by incorporating alabel detectable by, e.g., spectroscopic, photochemical, biochemical,immunochemical, chemical, or other techniques. To illustrate, usefullabels include radioisotopes, fluorescent dyes, electron-dense reagents,enzymes (as commonly used in ELISAs), biotin, or haptens and proteinsfor which antisera or monoclonal antibodies are available. Many of theseand other labels are described further herein and/or are otherwise knownin the art. One of skill in the art will recognize that, in certainembodiments, primer nucleic acids can also be used as probe nucleicacids.

A “subsequence” or “segment” refers to any portion of an entire nucleicacid sequence. A “substantially identical variant” in the context ofnucleic acids or polypeptides, refers to two or more sequences that haveat least 85%, typically at least 90%, more typically at least 95%nucleotide or sequence identity to one another when compared and alignedfor maximum correspondence, as measured using, e.g., a sequencecomparison algorithm or by visual inspection. The substantial identitygenerally exists over a region of the sequences that is at least about15 nucleotides or amino acids in length, more typically over a regionthat is at least about 20 nucleotides or amino acids in length, and evenmore typically the sequences are substantially identical over a regionof at least about 25 nucleotides or amino acids in length. In someembodiments, for example, the sequences are substantially identical overthe entire length of the nucleic acids or polypeptides being compared.The term “substitution” in the context of a nucleic acid sequence refersto an alteration in which at least one nucleotide of the nucleic acidsequence is replaced by a different nucleotide. The terms “targetsequence,” “target region,” and “target nucleic acid” refer to a regionof a nucleic acid, which is to be amplified, detected, or otherwiseanalyzed.

A “terminator nucleotide” refers to a nucleotide, which uponincorporation into a nucleic acid prevents further extension of thenucleic acid, e.g., by at least one nucleotide incorporatingbiocatalyst.

A “thermostable enzyme” refers to an enzyme that is stable to heat, isheat resistant and retains sufficient catalytic activity when subjectedto elevated temperatures for selected periods of time. For example, athermostable polymerase retains sufficient activity to effect subsequentprimer extension reactions when subjected to elevated temperatures forthe time necessary to effect denaturation of double-stranded nucleicacids. Heating conditions necessary for nucleic acid denaturation arewell known in the art and are exemplified in U.S. Pat. Nos. 4,683,202and 4,683,195, which are both incorporated by reference. As used herein,a thermostable polymerase is typically suitable for use in a temperaturecycling reaction such as the polymerase chain reaction (“PCR”). For athermostable polymerase, enzymatic activity refers to the catalysis ofthe combination of the nucleotides in the proper manner to form primerextension products that are complementary to a template nucleic acid(e.g., selected subsequences of Candidatus Liberibacter (LAS) genome).

A “quencher moiety” or “quencher” refers to a moiety that reduces and/oris capable of reducing the detectable emission of radiation, e.g.,fluorescent or luminescent radiation, from a source that would otherwisehave emitted this radiation. A quencher typically reduces the detectableradiation emitted by the source by at least 50%, typically by at least80%, and more typically by at least 90%. Exemplary quenchers areprovided in, e.g., U.S. Pat. No. 6,465,175, entitled “OLIGONUCLEOTIDEPROBES BEARING QUENCHABLE FLUORESCENT LABELS, AND METHODS OF USETHEREOF,” which issued Oct. 15, 2002 to Horn et al., which isincorporated by reference.

The term “sample” refers to any substance containing or presumed tocontain one or more host and/or pathogen nucleic acids including, butnot limited to, tissue or fluid isolated from one or more subjects orindividuals, in vitro cell culture constituents, as well as clinicalsamples. Exemplary samples include bacteria, virus, plant, animal ormammal sample including blood, plasma, serum, urine, synovial fluid,seminal fluid, seminal plasma, prostatic fluid, vaginal fluid, cervicalfluid, uterine fluid, cervical scrapings, amniotic fluid, analscrapings, mucus, sputum, tissue, and the like.

The phrase “sample derived from a subject” refers to a sample obtainedfrom the subject, whether or not that sample undergoes one or moreprocessing steps (e.g., cell lysis, debris removal, stabilization, etc.)prior to analysis. To illustrate, samples can be derived from subjectsby scraping, venipuncture, swabbing, biopsy, or other techniques knownin the art.

A “sequencing reaction” refers to a reaction that includes, e.g., theuse of terminator nucleotides and which is designed to elucidate thesequence of nucleotides in a given nucleic acid. A “set” refers to acollection of at least two things. For example, a set may include 2, 3,4, 5, 10, 20, 50, 100, 1,000 or other number of molecule or sequencetypes. For example, certain aspects of the invention include reactionmixtures having sets of amplicons. A “subset” refers to any portion of aset.

A “solid support” refers to a solid material that can be derivatizedwith, or otherwise attached to, a chemical moiety, such as anoligonucleotide probe or the like. Exemplary solid supports includeplates, beads, microbeads, tubes, fibers, whiskers, combs, hybridizationchips (including microarray substrates, such as those used in GeneChip™probe arrays (Affymetrix, Inc., Santa Clara, Calif., USA) and the like),membranes, single crystals, ceramic layers, self-assembling monolayers,and the like.

A “subject” refers to an organism. Typically, the organism could be abacteria organism, viral organism, plant organism, animal or a mammalianorganism including a human organism. In certain embodiments, forexample, a subject is a plant or tree suspected of having LAS or citrusgreening infection or disease, such as HLB.

The invention provides a novel pathogenesis model illustrating archaicphage infection of HLB. More specifically, the invention provides thatunder stress and/or in the presence of inducers or modulators of plantor bacterial origin, the phage genes attach/adsorb to the bacterialtargeted genes result in peptidoglycan destruction in preparation of“burst”, as well as phage protein production and phage assembly (“BurstMarker”) followed by phage release in the lytic stage where thebacterial components, such as phage particles, bacterial cell wall,starch, remnant peptidoglycan and cellular contents, are releasedsimultaneously in Lytic Phage Burst (LPB), resulting in “greeningeffect” where downstream phloem and/or leaves are clogged and dried upin a “river bed” corky appearance, and transpiration is halted for thissection of tree independent of other systemic nutrient routes (see FIGS.1, 3, 4, and 5).

FIG. 1 illustrates a novel pathogenesis model. This model illustratesthat under stress (103), e.g., in the presence of inducers or modulatorsof plant or bacterial origin (104), the phage genes (101) attach/adsorbto the bacterial targeted genes (102) result in phage protein productionand phage assembly (“Burst Marker”) (106) and peptidoglycan (105)destruction in preparation of “burst” followed by phage release in the“lytic stage” (107) where the bacterial components are released. Incertain embodiments, the released bacterial starch glucose molecules(108) clog phloem of stems & leaves (109), in a manner similar to humanheart disease, thus, blocking normal transpiration.

FIG. 2 illustrates normal pores in angiosperm sieve tube elements andcompanion cells. The cell wall (201), plastid (202), and vacuole (203)of the companion cell (200), and the mitochondrion (301), degeneratingSER (302), plastid (303), cell wall (304), P-protein (305), degeneratetonoplast (306), as well as large plasmodesmate (307), sieve area (308),and lumen (309) of the sieve element (300) are illustrated in thefigure. The figure also illustrates normal transpiration flow in thedirection of the arrow through these normal pores.

FIG. 3 illustrates HLB flagellated in angiosperm sieve tube elements andcompanion cells wherein biofilm (401) is formed when bacteria are understress (inducer) (403) resulting in HLB (402), forming sieve plate(404), thus, leading to blocking the normal transpiration, as shown inFIGS. 4 and 5.

FIG. 4 illustrates blocked pores of the sieve plate where phageparticles, bacterial cell wall, starch, remnant peptidoglycan and/orcellular contents are released simultaneously in Lytic Phage Burst(LPB).

FIG. 5 illustrates the “Greening Effect” where downstream phloem (500)is dried up in a “river bed” corky appearance. Transpiration is haltedfor this section of tree independent of other systemic nutrient routes.

Certain embodiments of the invention provide phage production in HLB.Functional holin activity of the SC1_gp110 gene confirms the annotation.In certain embodiments, the invention provides that expression analysesrevealed much higher levels of expression of phage late genes,particularly the holin, in periwinkle as compared to citrus, indicatinga phage response specific to periwinkle. In other embodiments, thesignificant increase in holin mRNA levels in citrus, 12 to 24 hoursafter being detached, regardless of temperature, indicates a potentiallysis triggering signal in citrus. In yet other embodiments, the holinpromoter region was developed into a reporter gene construct that isuseful for monitoring lytic activation by potential inducers in citrus,and it was demonstrated that the mode of action of thermal therapy (heatcuring of Las) in infected citrus does not seem to be connected to phageinduction.

The invention further provides a selective detection of one or morepathogens in a subject based on molecular biology techniques andreagents. In particular, based on new detection strategies utilizingquantitative load test with at least two (2) or three (3) targets fromat least two different organisms that are pathogenically tied togetherto derive a pathogenic ratio or load or index used in diagnosis,prognosis and/or treatment of any disease involving virus or phage in aratio to host (e.g., bacteria or plant) in plants and/or animals ormammals including humans. More specifically, based on the methods andreagents described herein, Candidatus Liberibacter asiaticus (LAS) orcitrus greening infections can be diagnosed by utilizing a phage DNA orRNA quantification as a pathogenic ratio of pathogenesis with its host,i.e., the LAS bacteria genome, for reporting pathogenesis and treatmentstrategy with clinical utility. In addition to detection methods andreaction mixtures, the invention also provides kits and systems fordetecting these pathogenic agents, as well as the use of the systems oran in vitro cell culture model comprising a phage lytic cycle repressorfor drug screening for treating and preventing HLB and citrus greening.Moreover, the invention provides methods and compositions of treatmentand prevention of HLB and citrus greening using any antiviral agentsincluding, but not limited to, nucleoside, nucleotide, ribonucleoside oranalogs thereof, with or without a nucleotide pool reducer, such ashydroxyurea. Furthermore, the invention provides that the treatment orprevention composition can further comprise a surfactant or penetrant tofacilitate drug delivery to the target location of the subject forbetter targeted treatment.

Pathogen Load or Pathogen Index for Diagnostic and Prognosis ofPathogenesis and/or Citrus Greening Blight, such HLB or LAS

The invention provides a novel pathogenesis model for diagnostic,prognostic, and treatment methods in a biological sample.

In certain embodiments, the invention provides a method for detectingpathogenesis in a biological sample, comprising: a) quantifying theamount of a nucleic acid in the sample specific for a first organismthat is a pathogen associated with a second organism that in tandemthese two organisms affects a host organism to determine a pathogenquantitative measurement, b) quantifying the amount of a nucleic acid inthe sample specific for the host organism response to pathogens todetermine a multiplex quantitation for targets associated with thepathogenesis of the disease; and c) calculating the ratio of the amountof dual pathogen targets relative to the amount of host quantitativemeasures, wherein said ratio provides a pathogenic index for detectingpathogenesis. In certain embodiments, a greater pathogenic indexindicates a poorer prognosis and can be used for drug treatment efficacyand drug screening.

In certain embodiments, the quantification of the nucleic acid in thesample specific for the first organism is performed by contacting thenucleic acid from the sample with a first set of oligonucleotide primersin a nucleic acid amplification reaction, the first set ofoligonucleotides being at least partially complementary to the nucleicacid of the first organism, and wherein the quantification of thenucleic acid in the sample specific for the second organism is performedby contacting the nucleic acid from the sample with a second set ofoligonucleotide primers in a nucleic acid amplification reaction, thesecond set of oligonucleotides being at least partially complementary tothe nucleic acid of the second organism, and further comprisingdetecting amplicons from the nucleic acid amplification reactions, andwherein the quantification of the nucleic acid in the sample specificfor the third host organism is performed by contacting the nucleic acidfrom the sample with a third set of oligonucleotide primers in a nucleicacid amplification reaction, the third set of oligonucleotides being atleast partially complementary to the nucleic acid of the third organism,and further comprising detecting amplicons from the nucleic acidamplification reactions, wherein the amplification reactions can occurin the same container, and calculating the ratio of the amount ofpathogen ratio relative to the amount of host load based on the relativenumber of amplicons to derive the pathogenic index or quartile. As usedherein, the pathogen organism is a virus or bacteria, and the hostorganism is a bacteria, plant or animal. When the host organism is abacteria, the host may also reside within or in association with anotherhost, such as a plant or animal, e.g., human. In certain embodiments,the pathogenesis is citrus green blighting or Huanglongbin (HLB), thepathogen or the first organism can be a bacterio-phage or virus, and thesecond organism is Candidatus Liberibacter asiaticus (LAS) bacteria andthe host is a wide array of fruiting and non-fruiting plants. Thepathogenic index may also be derived from a further correlation with anyinducer or factor gene pathogenically tied together with the dualpathogen to host relationship. In certain embodiments, the secondorganism is a lytic bacteriophage, including but not limited to, SC1,SC2, and SC3 lytic phage.

The nucleic acid quantified in the invention method includes, but is notlimited to, RNA or DNA. In certain embodiments, the phage RNA and thebacterial DNA are quantified, and a ratio of phage RNA to bacterial DNAis calculated to derive a pathogen index or quartile. The nucleic acidcan be amplified by any conventional nucleic acid amplificationreactions for quantitation (e.g., qPCR) including, but not limited tostandard polymerase chain reaction (PCR), real-time PCR (e.g., Roche,HIV-1 Cobas Taqman system; Abbot HIV-1 M2000 System), recombinasepolymerase amplification (RPA), helicase-dependent amplification (HAD),loop mediate isothermal amplification (LAMP), and nicking enzymeamplification reaction (NEAR). Details describing qPCR and/or real-timePCR can be found in U.S. Pat. Nos. 5,972,716; 6,800,452; 6,746,864;7,427,380; 7,238,321; and 8,058,054, and Tatineni et al. (2008,Phytopathology 98(5):592-99), the entire contents of each of which areincorporated by reference herein. Furthermore, the detailed descriptionsof the RPA amplification are provided for instance in U.S. Pat. Nos.7,485,428; 7,666,598; 7,763,427; and 7,759,061, the entire contents ofeach of which are incorporated by reference herein; the detaileddescriptions of the HAD amplification are provided for instance in U.S.Pat. Nos. 7,829,284; 7,282,328;and 7,662,594, the entire contents ofeach of which are incorporated by reference herein; and the detaileddescriptions of the LAMP amplification are provided for instance in U.S.Pat. Nos. 7,851,186; 7,745,135; and 6,974,670, the entire contents ofeach of which are incorporated by reference herein.

In certain embodiments, the invention provides a kit for detectingcitrus greening blight in a sample, comprising: a) a first set ofoligonucleotides being at least partially complementary to a nucleicacid of a first pathogen organism in the sample that is pathogenicallyassociated with citrus greening blight, b) a second set ofoligonucleotides being at least partially complementary to a nucleicacid of a second organism that is a host to the pathogen organism in thesame sample, c) reagents for nucleic acid amplification reactions withthe first and second sets of oligonucleotides; d) instructions forconducting the nucleic acid amplification reactions and detecting one ormore amplicons thereof; and e) instructions for calculating a ratio ofthe amplicons wherein said ratio provides a pathogenic index fordetecting, prognosis, and treatment for citrus greening blight.

Typically, at least one of the nucleic acid detection reagents comprisesat least one label and/or at least one quencher moiety. To illustrate,the label optionally comprises a fluorescent dye, a weakly fluorescentlabel, a non-fluorescent label, a colorimetric label, a chemiluminescentlabel, a bioluminescent label, an antibody, an antigen, biotin, ahapten, a mass-modifying group, a radioisotope, an enzyme, or the like.

The nucleic acid detection reagents of the invention are provided invarious formats. In some embodiments, for example, at least one of thenucleic acid detection reagents is in solution. In other embodiments, asolid support comprises at least one of the nucleic acid detectionreagents. In these embodiments, the nucleic acid detection reagents arenon-covalently or covalently attached to the solid support. Exemplarysolid supports utilized in these embodiments are optionally selectedfrom, e.g., a plate, a microwell plate, a bead, a microbead (e.g., amagnetic microbead, etc), a tube (e.g., a microtube, etc.), a fiber, awhisker, a comb, a hybridization chip, a membrane, a single crystal, aceramic layer, a self-assembling monolayer, and the like.

To further illustrate, the nucleic acid detection reagents areoptionally conjugated with biotin or a biotin derivative and the solidsupport is optionally conjugated with avidin or an avidin derivative, orstreptavidin or a streptavidin derivative. In some embodiments, a linkerattaches the nucleic acid detection reagents to the solid support. Thelinker is typically selected from, e.g., an oligopeptide, anoligonucleotide, an oligopolyamide, an oligoethyleneglycerol, anoligoacrylamide, an alkyl chain, and the like. Optionally, a cleavableattachment attaches the nucleic acid detection reagents to the solidsupport. The cleavable attachment is generally cleavable by, e.g., heat,an enzyme, a chemical agent, electromagnetic radiation, etc.

The kit also includes one or more of: a set of instructions forcontacting the nucleic acid detection reagents with nucleic acids from asample or amplicons thereof and detecting binding between the nucleicacid detection reagents and the target nucleic acids, if any, or atleast one container for packaging the nucleic acid detection reagentsand the set of instructions. Exemplary solid supports include in thekits of the invention are optionally selected from, e.g., a plate, amicrowell plate, a bead, a microbead, a tube (e.g., a microtube, etc.),a fiber, a whisker, a comb, a hybridization chip, a membrane, a singlecrystal, a ceramic layer, a self-assembling monolayer, or the like.

Diagnostic, Prognostic, and Treatment Report:

The invention further provides a report comprising a report algorithmcorrelated with the pathogenic index providing clinical utility as atreatment strategy. The TRUGENE® HIV-1 Genotyping Kit (Visible Genetics,Siemens) is exemplary of a report algorithm with clinical utility usedas a treatment strategy. In particular, the TRUGENE HIV-1 GenotypingAssay is based upon several processes culminating in a data analysisstep, wherein gene sequences are analyzed by a software system toprovide a TRUGENE HIV-1 Resistance Report that is useful for thetherapeutic management of HIV in a patient, also see U.S. Pat. Nos.6,830,887; 6,653,107; 6,265,152; 6,007,983; 5,795,722; 5,834,189; and5,545,527, the entire contents of each of which are incorporated byreference herein.

In some embodiments, the invention method comprises a step ofquantifying the amount of a nucleic acid in the sample specific for hostbacteria, phage pathogen, and an inducer of the pathogen organism todetermine a host load, a pathogen load, and an inducer load; and whereinthe pathogenic index, host or pathogen load, and/or the inducer load arecorrelated with the report index for determining prognosis and providingclinical utility as a treatment strategy. The invention method furtherprovides that multiple unique nucleic acids specific to the pathogenorganism, the host organism or the inducer are quantified and correlatedin the report index.

Cultivation of Citrus Greening Blight for Screening a TreatmentTherapeutic or Research

The invention further provides a method for screening a candidatetherapeutic agent for inhibition or prevention of citrus greeningblight. Cultivation of ‘Candidatus Liberibacter asiaticus’, ‘Ca. L.africanus’ and ‘Ca. L. americanus’ associated with Huanglongbing isreported in Sechler et al. (2009, Phytopathology 99 (5):480-86), theentire content of which is incorporated by reference herein. Theinvention method comprises the steps of providing a culture ofCandidatus Liberibacter with a bacteriophage having a repressed phagelytic cycle; combining the culture with a candidate therapeutic agentcandidate; and detecting inhibition of growth of the CandidatusLiberibacter culture indicating that the candidate therapeutic agentinhibits or prevents citrus greening blight. In certain embodiments, thebacteriophage is a lytic phage, including but not limited to, SC1, SC2,and SC3 lytic phage, and wherein the phage lytic cycle is repressed orgenetically deleted. In some embodiment, the citrus greening blightbacteria, such as the HLB bacteria Candidatus Liberibacter is culturedin a culture media containing a repressor to phage lytic cycle of abacteriophage. The invention encompasses any cell culture media andsystem suitable for growing any bacteria cultures. The invention alsoencompasses any phage lytic cycle repressor or inhibitor, now known orlater developed in the art, including but not limited to SC1 repressorsSC1_gp110 (holin), SC1_gp025, SC1_gp-95, SC2_gp100, C1 or phage lambdarepressor (e.g., phage 434, P22), see Exploiting the Las and Lam phagefor potential control of HLB, Citrus Advanced Technology Program, finalreport submitted on Aug. 7, 2013 by Citrus Research and DevelopmentFoundation, the entire report is incorporated by reference herein.

Prevention and Treatment of Bacteriophage or Viral Infection

The invention also provides a method for treating and/or preventingbacteriophage or viral infection in bacteria, plant or animalindividual, said method comprising administering to the individual inneed a composition comprising an effective amount of an agent selectedfrom the group consisting of nucleoside, non-nucleoside, nucleotide,non-nucleotide, ribonucleoside, and a ribonucleoside analog thatselectively inhibits viral replication. Nucleosides can bephosphorylated by specific kinases in the cell on the sugar's primaryalcohol group (—CH₂—OH), producing nucleotides, which are the molecularbuilding-blocks of DNA and RNA. Nucleosides can be produced by de novosynthesis pathways, in particular in the liver, but they are moreabundantly supplied via ingestion and digestion of nucleic acids in thediet, whereby nucleotidases break down nucleotides (such as thethymidine monophosphate) into nucleosides (such as thymidine) andphosphate. The nucleosides, in turn, are subsequently broken down in thelumen of the digestive system by nucleosidases into nucleobases andribose or deoxyribose. In addition, nucleotides can be broken downinside the cell into nitrogenous bases, and ribose-1-phosphate ordeoxyribose-1-phosphate.

Several nucleoside analogues are also used as antiviral or anticanceragents. Nucleoside analogues are molecules that act like nucleosides inDNA synthesis. They include a range of antiviral products used toprevent viral replication in infected cells. The most commonly used isacyclovir (ACV). The use of nucleoside, non-nucleoside, nucleotide,non-nucleotide, ribonucleoside, a ribonucleoside analog, and anucleotide pool reducer are known in the art for treatment of viralinfection, such as for example, the treatment of human immunodeficiencyviruses. The viral polymerase incorporates these compounds withnon-canonical bases. These compounds are activated in the cells by beingconverted into nucleotides. They are administered as nucleosides sincecharged nucleotides cannot easily cross cell membranes.

Exemplary nucleoside analogue drugs include, but not limited todeoxyadenosine analogues (e.g., DIDANOSINE (ddI)(HIV) and VIDARABINE(chemotherapy)), deoxycytidine analogues (e.g., CYTARABINE(chemotherapy), EMTRICITABINE (FTC)(HIV), LAMIVUDINE (3TC)(HIV,hepatitis B), and ZALCITABINE (ddC)(HIV)), deoxyguanosine analogues(e.g., ABACAVIR (HIV) and Entecavir (hepatitis B)), (deoxy-)thymidineanalogues (e.g., STAVUDINE (d4T), TELBIVUDINE (hepatitis B), ZIDOVUDINE(azidothymidine, or AZT)(HIV)), and deoxyuridine analogues (e.g.,Idoxuridine and Trifluridine).

The invention encompasses any nucleoside or nucleoside analogs, nowknown or later developed in the art that are capable of treating a viralinfection. Exemplary nucleoside antiretroviral agents include, but notlimited to, ZIDOVUDINE® (ZDV, AZT), EPIVIR® (3TC, lamivudine), EMTRIVA®(FTC, emtricitabine), VIREAD® (tenofovir disoproxil fumarate, TDF),ZIAGEN® (abacavir sulfate, ABC), CARBOVIR® (CBV), Racivir [RCV,(±)-β-2′,3′-dideoxy-5-fluoro-3′-thiacytidine], DEXELVUCITABINE®(β-D-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine, reverset, RVT,D-D4FC, DFC), AMDOXOVIR® [AMDX, (−)-β-D-2,6-diaminopurine dioxolane,DAPD], 9-(beta-D-1,3-Dioxolan-4-yl)guanine (DXG), AVX754 [SPD-754,(−)dOTC, (−)-2′-deoxy-3′-oxa-4′-thiocytidine]. Non-nucleoside reversetranscriptase inhibitors (NNRTIs) represent another class of drugs thatmay be used in the present invention, and may be selected from NNRTIssuch as are used for treating HIV infections, such as efavirenz(SUSTIVA®), rilpivirine (EDURANT®), etravirine (INTELENCE®), delavirdine(RESCRIPTOR®), nivirapine (VIRAMUNE®)and lersivirine. See also U.S. Pat.Nos. 8,415,321; 8,168,583; 7,635,690; 7,402,588; 7,115,584; 6,949,521;and 5,990,093, the entire contents of each of which are incorporated byreference herein.

The amount of a nucleoside or analog thereof that may be used alone orin combination with a pharmaceutically acceptable carrier to produce asingle dosage form will vary depending upon the host treated and theparticular mode of administration. It will be appreciated by thoseskilled in the art that the unit content of a nucleoside or analogthereof contained in an individual dose of each dosage form need not initself constitute an effective amount, as the necessary effective amountcould be reached by administration of a number of individual doses. Theselection of dosage depends upon the dosage form utilized, the conditionbeing treated, and the particular purpose to be achieved according tothe determination of those skilled in the art.

The dosage regime for treating or preventing bacteriophage or viralinfection in bacteria, plant or animal individual with a nucleoside oranalog thereof of the invention is selected in accordance with a varietyof factors, including the type, age, weight, sex, diet and medicalcondition of the subject, the route of administration, pharmacologicalconsiderations such as activity, efficacy, pharmacokinetic andtoxicology profiles of the particular compound employed, whether anucleoside delivery system is utilized and whether the nucleoside isadministered as a pro-drug or part of a drug combination. Thus, thedosage regime actually employed may vary widely from subject to subject.

Any antiviral agent including the nucleoside or analog thereof used inthe present invention can be synthesized chemically and/or produced byany suitable mythology and/or technology known to those skilled in theart, and can be formulated by known methods for administration to asubject using several suitable routes including but not limited to,systemic or local administration depending on the subject being treatedor administered. The individual antiviral agent may also be administeredin combination with one or more other antiviral agent and/or togetherwith other biologically active or biologically inert agents. Suchbiologically active or inert agents may be in fluid or mechanicalcommunication with the antiviral agent or attached to the antiviral byionic, covalent, Van der Waals, hydrophobic, hydrophillic or otherphysical forces.

The antiviral agent, such as nucleoside or analog thereof, of thepresent invention may be formulated by any conventional manner using oneor more pharmaceutically acceptable carriers and/or excipients. Theantiviral agent of the invention may take the form of charged, neutraland/or other pharmaceutically acceptable salt forms. Examples ofpharmaceutically acceptable carriers include, but are not limited to,those described in REMINGTON'S PHARMACEUTICAL SCIENCES (A. R. Gennaro,Ed.), 20th edition, Williams & Wilkins PA, USA (2000).

The nucleoside or analog thereof may also take the form of solutions,suspensions, emulsions, tablets, pills, capsules, powders, controlled-or sustained-release formulations and the like. Such formulations willcontain an effective amount of the nucleoside or analog thereofpreferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

An effective amount of an antiviral agent (e.g., nucleoside or analogthereof) relates generally to the amount needed to achieve a preventiveor therapeutic objective, administration rate, and depletion ormetabolic rate of the antiviral agent from a subject. Common ranges foreffective doses, as well as dosing frequencies may vary depending on thefactors discussed above.

In some embodiments, the treatment and/or prevention method may alsoinclude a nucleotide pool reducer. In one aspect the nucleotide poolreducer is hydroxyurea or hydroxycarbamide. While not intending to bebound by theory, hydroxyurea has been shown to inhibit the de novosynthesis of deoxyribonucleotides, deplete deoxyribonucleosidetriphosphate pools and starve host cell and viral replication ofprecursor molecules, such as described in Hendricks and Matthews, 1998,and which is incorporated by reference herein.

In another aspect, the method for treating citrus greening blightincludes administering a composition comprising a surfactant orpenetrant for enhancing delivery of the composition. The term surfactantor penetrant means one or more compounds that improve and/or acceleratethe delivery of the composition. In certain embodiments, the inventioncomposition comprises a penetrant under a trade name Atomic Grow,manufactured by NanoCanopy. The invention encompasses any othersurfactant or penetrant, now known or later developed in the art, forthe same purposes. Various administration strategies for delivery toplants are well-known in the art.

Each of the publications referred to herein is hereby incorporated byreference in its entirety, for all purposes.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention.

1. A method for detecting pathogenesis in a biological sample,comprising: a) quantifying the amount of a nucleic acid in the samplespecific for a first organism that is a pathogen associated with asecond organism that in tandem the first and second organisms affect ahost organism to determine a pathogen quantitative measurement, b)quantifying the amount of a nucleic acid in the sample specific for thehost organism response to pathogen to determine a multiplex quantitationfor targets associated with the pathogen of a disease; and c)calculating the ratio of the amount of dual pathogen targets relative tothe amount of host quantitative measures, wherein said ratio provides apathogen index for detecting pathogenesis.
 2. The method of claim 1,wherein said pathogen index indicates a prognosis of the pathogenesis orcan be used for drug treatment efficacy and drug screening.
 3. Themethod of claim 1, wherein the quantification of the nucleic acid in thesample specific for the first organism is performed by contacting thenucleic acid from the sample with a first set of oligonucleotide primersin a nucleic acid amplification reaction, the first set ofoligonucleotides being at least partially complementary to the nucleicacid of the first organism, and wherein the quantification of thenucleic acid in the sample specific for the second organism is performedby contacting the nucleic acid from the sample with a second set ofoligonucleotide primers in a nucleic acid amplification reaction, thesecond set of oligonucleotides being at least partially complementary tothe nucleic acid of the second organism, and wherein the quantificationof the nucleic acid in the sample specific for the third host organismis performed by contacting the nucleic acid from the sample with a thirdset of oligonucleotide primers in a nucleic acid amplification reaction,the third set of oligonucleotides being at least partially complementaryto the nucleic acid of the third organism, and further comprisingdetecting amplicons from the nucleic acid amplification reactions,further comprising detecting amplicons from the nucleic acidamplification reactions, wherein the amplification reactions occur inthe same container, and calculating the ratio of the amount of pathogenratio relative to the amount of host load based on the relative numberof amplicons to derive the pathogenic index or quartile.
 4. The methodof claim 1, wherein said pathogen organism is a virus or bacteria. 5.The method of claim 1, wherein said host organism is a bacteria, plantor animal.
 6. The method of claim 1, wherein said pathogenesis is citrusgreen blighting or Huanglongbin (HLB).
 7. The method of claim 6, whereinsaid host organism is Candidatus Liberibacter asiaticus (LAS).
 8. Themethod of claim 7, wherein said pathogen organism is selected from agroup consisting of a phage, virus, and any inducer or factor genepathogenically tied together with LAS.
 9. The method of claim 8, whereinsaid second organism is a bacteriophage.
 10. The method of claim 9,wherein said bacteriophage is lytic phage selected from the groupconsisting of SC1, SC2, and SC3 lytic phage.
 11. The method of claim 1,wherein said nucleic acid is RNA or DNA.
 12. The method of claim 3,wherein said nucleic acid amplification reaction is real-time PCR,recombinase polymerase amplification (RPA), helicase-dependentamplification (HAD), loop mediate isothermal amplification (LAMP), andnicking enzyme amplification reaction (NEAR).
 13. The method of claim 1,further comprising a report algorithm or index correlated with thepathogen index or quartile providing clinical utility as a treatmentstrategy or drug screening.
 14. The method of claim 13, furthercomprising a step of quantifying the amount of a nucleic acid in thesample specific for an inducer of the pathogen organism to determine aninducer load; and wherein the pathogen index and the inducer load arecorrelated with the report index for determining prognosis and providingclinical utility as a treatment strategy or drug screening.
 15. Themethod of claim 14, wherein multiple unique nucleic acids specific tothe pathogen organisms, the host organism or the inducer are quantifiedand correlated in the report index.
 16. A kit for detecting pathogenesisin a sample, comprising: a) at least a first set of oligonucleotidesbeing at least partially complementary to a nucleic acid of a firstpathogen organism that is pathogenically associated with a secondorganism that in tandem these two organisms affects a host organism todetermine a pathogen quantitative measurement, b) at least a second setof oligonucleotides being at least partially complementary to a nucleicacid of the host organism response to pathogen of a disease, c) reagentsfor nucleic acid amplification reactions with the first and second setsof oligonucleotides; d) instructions for conducting the nucleic acidamplification reactions and detecting one or more amplicons thereof; ande) instructions for calculating a ratio of the amplicons or the amountof dual pathogen targets relative to the amount of host quantitativemeasures, wherein said ratio provides a pathogen index for detecting,prognosis, drug screening, and treatment strategy or efficacy for thepathogenesis.
 17. The kit of claim 16, wherein said pathogenesis iscitrus greening blight or HLB.
 18. A method for screening a candidatetherapeutic agent for inhibition or prevention of citrus greening blight(HLB), comprising providing a culture of Candidatus Liberibacter with abacteriophage having a repressed phage lytic cycle; combining theculture with a candidate therapeutic agent candidate; and detectinginhibition of growth of the Candidatus Liberibacter culture indicatingthat the candidate therapeutic agent inhibits or prevents citrusgreening blight (HLB).
 19. The method of claim 18, wherein said isbacteriophage is a lytic phage selected from the group consisting ofSC1, SC2, and SC3 lytic phage.
 20. The method of claim 18, wherein thephage lytic cycle is repressed or genetically deleted.
 21. A method fortreating bacteriophage infection in bacteria, plant or animal individualcomprising administering to the individual in need a compositioncomprising an effective amount of an agent selected from the groupconsisting of nucleoside, non-nucleoside, nucleotide, non-nucleotide,ribonucleoside, and a ribonucleoside analog that selectively inhibitsviral replication.
 22. A method for treating viral infection in a plantcomprising administering to the plant in need a composition comprisingan effective amount of an agent selected from the group consisting ofnucleoside, non-nucleoside, nucleotide, non-nucleotide, ribonucleoside,and a ribonucleoside analog that selectively inhibits viral replication.23. A method for treating citrus greening blight comprisingadministering to a citrus tree in need a composition comprising aneffective amount of an agent selected from the group consisting ofnucleoside, non-nucleoside, nucleotide, non-nucleotide, ribonucleoside,and a ribonucleoside analog that selectively inhibits viral replication.24. The method of claim 23, wherein said composition further compriseshydroxyurea or another nucleotide pool reducer in an effective amount.25. The method of claim 23, wherein said composition further comprises asurfactant or penetrant for enhancing delivery of said composition to atargeted location of the subject.
 26. A method for preventing citrusgreening blight comprising administering to a citrus tree in need acomposition comprising an effective amount of an agent selected from thegroup consisting of nucleoside, non-nucleoside, nucleotide,non-nucleotide, ribonucleoside, and a ribonucleoside analog thatselectively inhibits viral replication.
 27. The method of claim 26,wherein said composition further comprises hydroxyurea or anothernucleotide pool reducer in an effective amount.
 28. The method of claim25, wherein said composition further comprises a surfactant or penetrantfor enhancing delivery of said composition to a targeted location of thesubject.